Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Acute toxic effects are noted.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD and Japanese testing guidelines in compliance with GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test animals: Thirty males and 30 females of Crj:CD (SD) strain rats [SPF] were purchased from Japan Charles River Co. Ltd., (Atsugi-shi, Kanagawa) at the age of 5 weeks old and used in the study.
After inspection, the animals were acclimatized to the test environment and the administration of test substance was started at the age of 6 weeks old.
The animals were stratified by the body weights, and allocated to each test group by randomization procedure.
The animals were identified by the marking method with saturated picric acid in ethanol and attaching the labels specified with animal identification number (Animal ID-No.). The body weights at the start of administration were 145-164 g in male and 116-145 g in female.

Reason for test system selection: The species was selected considering the resistance to infectious diseases, genetic stability and others.

Animal husbandry: The animals were housed in an animal room (W 3.6 x D 10.0 x H 2.5m, 90 m3), the target environmental conditions were 23 ± 2°C of temperature, 55 ± 10% of relative humidity, 20 times per hour of ventilation frequency and 12-hour lighting (150-300 lux, lighting at 7: 00 a.m. and turn off at 7:00 p.m.).
Five animals were housed in each metal-mesh cage (W 21.5 x D 27.5 x H 19.5 cm, space: 11529 cm3). Cages were set on a water-flush breeding instrument (W 745.0 x D 50.0 x H 182.0 cm) supplied by Tokyo Giken Service Co. Ltd (Fuchu-shi, Tokyo).
Cages and food supplier were exchanged once a week.
The food used was solid food MF supplied by Oriental Yeast Co., Ltd. (Chuo-ku, Tokyo). The food was given to the animals ad libitum.
In addition, the animals were allowed free access to drinking water supplied by automatic water supply nozzles.
There were no environmental deviations which might influence the reliability of the study data during the duration of observation including the acclimatization period.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Reasons for the dose selection: In the preliminary study 500, 1000 and 2000 mg/kg were orally administered to 3 males and 3 females each. As a result, 1 male and 1 female in 1000 mg/kg group died, and all animals in 2000 mg/kg group died. No animal died in 500 mg/kg group.
From the above results, the five dose levels were selected in the main study based on the geometric ratio of 1.25 as 819-2000 mg/kg in male and 655-1600 mg/kg in female.

Preparation of dosing solution: Appropriate amount of the test substance was weighed with an electronic balance and dissolved in corn oil (Nacalai Tesque Inc., Kyoto-shi, Kyoto). The concentration of solutions were 13.1, 16.4, 20.5, 25.6, 32.0 and 40.0 (w/v)% respectively for 655, 819, 1024, 1280, 1600 and 2000 mg/kg groups.
Doses:
Dose levels were 819, 1024, 1280, 1600 and 2000 mg/kg in male, 655, 819, 1024, 1280 and 1600 mg/kg in female.
No. of animals per sex per dose:
5 male and 5 female animals per dose group.
Control animals:
no
Details on study design:
Administration procedure: The administration route of the test substance was determined as oral route. The test substance dissolved in corn oil was administered by gavage using a steel cannula into the stomach of animals which were starved for 16 hours before the administration.
The volume to administer to each animal was determined as 0.5 mL per 100 g of body weight.
Food was given to the animals at approximately 3 hours after the administration of test substance.

Observations: Observations of toxic signs and mortality were conducted in the interval of an hour until 6 hours after the administration, and then, twice a day, in the morning and afternoon (morning only on holidays) until 14 days after the administration. Toxic signs observed were recorded in the sheets for observation findings.

Body weight: Animals were weighed immediately before the administration and at 7 and 14 days after the administration.
In addition, dead animals were weighed at the discovery of death.

Pathological examination
Macroscopic observation: Dead animals during the observation period were autopsied on the discovery, and survived animals were euthanized by exsanguination under ether anesthesia and autopsied on the day of observation end. Macroscopic abnormalities were recorded on the sheet for the record of macroscopic findings.

Histopathological examination: The organs or tissues with abnormalities were preserved in 10% neutral buffered formalin, and parts from them were processed for histopathological examination. Histological slides were prepared and examined under microscope, and the pathological findings were recorded for the type and severity of changes.

Disposal of remained animals: Remained animals were euthanized by carbon dioxide.
Statistics:
Calculation method of 50% lethal dose (LD50): LD50 was calculated by the method of Litchfield-Wilcoxon method (1949) from the mortalities at 14 days after the administration.
Preliminary study:
In the preliminary study 500, 1000 and 2000 mg/kg were orally administered to 3 males and 3 females each. As a result, 1 male and 1 female in 1000 mg/kg group died, and all animals in 2000 mg/kg group died. No animal died in 500 mg/kg group.
Sex:
male
Dose descriptor:
LD50
Effect level:
910 mg/kg bw
Based on:
test mat.
95% CL:
> 570 - < 1 452
Sex:
female
Dose descriptor:
LD50
Effect level:
972 mg/kg bw
Based on:
test mat.
95% CL:
> 814 - < 1 162
Mortality:
Mortalities in the male groups administered with 819, 1024, 1280, 1600 and 2000 mg/kg were 0, 60, 60, 80 and 100%, respectively. In female, the mortalities in the groups of 655, 819, 1024, 1280 and 1600 mg/kg were 0, 20, 60, 100 and 100%, respectively.
Clinical signs:
other: Decrease in locomotive activity was seen at 1 hour until 5 days after the administration. In addition, prone position and hunchback position were observed from 1 hour until 4 days after the administration, and abnormal gait was seen from 6 hours until 3 d
Gross pathology:
Macroscopic findings: In dead animals, black patch in the thymus, retention of brownish urine in the urinary bladder, and black contents and white patch in the small intestine were observed both in male and female. In addition, hydrothorax, black patch in the urinary bladder, white patch in the forestomach, black patch in the glandular stomach, white contents in the small and large intestine and brown contents in the small intestine in female animal were observed.
In the survived animals, adhesion with the forestomach and the diaphragm was seen both in male and female at the end of observation period. In addition, yellow patch in the liver, adhesion with the forestomach and the liver, adhesion with the liver and the diaphragm or the retro-peritoneum, adhesion with the liver and the spleen, adhesion with the spleen and the kidney and thickening of the forestomach were seen in male, atrophy of the spleen was seen in female.

Histopathology findings: In the survived male animals at the end of observation period, severe ulceration and granulomatous inflammation, and hyperplasia of the squamous epithelium and granulomatous inflammation were observed in the forestomach, and granulomatous inflammation was seen in the liver.
Other findings:
Detailed clinical signs
Male:
In 819 mg/kg group, decrease in locomotive activity was seen in 2 rats out of 5 from 1 hour after the administration, and in all rats from 2 hours to 3 days after the administration. Prone position was seen in 1 rat from 1 hour and 3 rats from 4 hours, hunchback position was seen in 1 rat from 5 hours after the administration and these signs lasted until 6-24 hours after the administration. In addition, abnormal gait appeared in 2 rats at 6 hours and later after the administration and lasted until 2 days after the administration. These signs were not severe and no death was observed.
In 1024 mg/kg group, decrease in locomotive activity was seen in all of 5 rats from 1 hour after the administration, and the sign lasted until 3 days after the administration in survived animals. Prone position was observed in 4 rats from 4 hours after the administration and it lasted until 3 days after the administration when the animals died, and hunchback position was observed in 2 rats from 5 hours after the administration and the sign lasted until 2 days after the administration. In addition, abnormal gait was observed in 2 rats at 6 hours and later after the administration and it lasted until 2 days after the administration, and lateral position was seen in 1 rat at 6 hours and later and lasted until the death of this animal. In addition to these changes, hypothermia was seen in 1 rat at 6 hours and later after the administration, it was observed in 2 rats out of 4 on day 3. Brownish urine was observed in 1 rat out of 4 at 3 days after the administration. Death was observed during 6-24 hours after the administration and 2 rats on day 3.
In 1280 mg/kg group, decrease in locomotive activity was observed in all of 5 rats from 1 hour after the administration and this sign in survived animals lasted until 5 days after the administration. Prone position was observed in 1 rat from 2 hours after the administration, in all animals during 4-6 hours after the administration and lasted until 3 days after the administration. Abnormal gait was seen in 1 rat during 6-24 hours after the administration. In addition, lateral position and hunchback position were seen in 1 and 2 rats, respectively at 6 hours and later after the administration, and these signs lasted until 2 or 3 days after the administration when the animals died. Hypothermia was seen in 2 rats at 6 hours and later and in 3 rats on day 2, and it lasted until 3 days after the administration. Furthermore, brownish urine was seen in 2 out of 4 rats at 3 days after the administration. One rat died on 2 days and 2 rats died on 3 days after the administration.
In 1600 mg/kg group, decrease in locomotive activity was seen in all 5 rats at 1 hour and lasted until 4 days after the administration in survived animals. Prone position was seen in 1 rat at 2 hours and in 3 rats at 4 hours and later and lasted until 6 hours after the administration. Hunchback position was seen in 1 rat at 4 hours after the administration, and lasted until 3 days after the administration, lateral position was observed in 1 rat from 5 hours after the administration and in 4 rats at 6 hours and later, and lasted until 2 days after the administration when the animals died. In addition, abnormal gait was observed in 1 rat at 6 hours and later after the administration, lasted until 3 days after the administration, and hypothermia was observed in 4 rats at 6 hours and later, lasted until the animals died. Four animals died on 2 days after the administration.
In 2000 mg/kg group, decrease in locomotive activity was observed in all 5 rats from 1 hour after the administration and lasted for 6-24 hours or until day 4 when the animals died. Prone position appeared from 4 hour after the administration in 4 rats, and lasted until 2 days after the administration. Hunchback position was seen from 5 hours in 1 rat, lasted during 6-24 hours after the administration, and abnormal gait was observed in 2 rats from 6 hours and later, lasted until 2 days after the administration. Lateral position was seen in 1 rat from 6 hours and later and in 1 survived rat during 3 and 4 days after the administration. In addition, hypothermia was observed in 2 rats at 6 hours and later after the administration and lasted until the animals died. All of the animals showing these signs died from 6 hours to 4 days after the administration.
Female:
In 655 mg/kg group, decrease in locomotive activity was seen 2 out of 5 rats from 3 hours and in 3 rats from 4 hours to 3 days after the administration. Prone position was observed in 2 rats from 5 hours after the administration and lasted until 6-24 hours. In addition, abnormal gait was observed in 1 rat from 6 hours and in 3 rats at 2 days after the administration. These symptoms were not severe and no death was observed.
In 819 mg/kg group, decrease in locomotive activity was seen in 4 rats out of 5 from 1 hour after the administration and in 5 rats from 2 hours to 2 days after the administration, and lasted until 3 days after the administration in survived animals. Prone position was seen in 1 rat from 4 hours after the administration and lasted until 6 hours after the administration. Abnormal gait and hunchback position were observed in 2 animals from 6 hours and later and lasted until 2 days after the administration. In addition, lateral position and hypothermia were seen in 1 animal from 6 hours after the administration and lasted until 2 days after the administration when the animal died. One death was noted on 2 days after the administration.
In 1024 mg/kg group, decrease in locomotive activity was seen in all 5 rats from 1 hour and lasted until 3 days after the administration in survived animals. Prone position was seen in 1 rat from 2 hours and in 4 rats from 4-6 hours after the administration. Abnormal gait was observed in 1 rat during 6-24 hours after the administration. In addition, lateral position and hypothermia were seen in 3 rats from 6 hours and lasted until 2 days after the administration when the animals died.
Hunchback position was seen in 1 rat from 6 hours and lasted until 2 day after the administration. Furthermore, brownish urine was noted in 1 rat on 2 days after the administration. 3 animals died on 2 days after the administration.
In 1280 mg/kg group, decrease in locomotive activity was observed in all 5 rats from 1 hour and lasted during 6-24 hours or until 2 days after the administration when the animals died. Prone position was seen in 3 rats from 4 hours after the administration and lasted until the animals died. Hunchback position was seen in 1 rat from 5 hours and lasted until 6 hours after the administration. In addition, lateral position was seen in 1 rat from 6 hours after the administration and in 4 rats at 6 hours and later, hypothermia was seen in 4 rats at 6 hours and later after the administration and these symptoms lasted until the animals died. Furthermore, brownish urine was seen in 1 rat out of 4 at 2 days after the administration. All animals which showed these symptoms died from 6 hours to 2 days after the administration.
In 1600 mg/kg group, decrease in locomotive activity was seen in all 5 rats from 1 hour after the administration, and lasted for 6-24 hours or until 2 days after the administration when the animals died. Prone position was seen in 2 rats from 2 hours and in 4 rats from 4-6 hours, and lasted until 6-24 hours after the administration. Hunchback position was seen in 1 rat from 4 hours after the administration, and lasted until the animal died. In addition, lateral position and hypothermia were seen in 3 rats from 6 hours after the administration, and lasted until the animals died. All animals which showed these symptoms died from 6 hours to 2 days after the administration.

Table 1. Mortality

Sex

Group

Dose level (mg/kg)

Number of animals

Number of deaths on the day

Mortality (%)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Male

1

819

5

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2

1024

5

1

0

2

0

0

0

0

0

0

0

0

0

0

0

60

3

1280

5

0

1

2

0

0

0

0

0

0

0

0

0

0

0

60

4

1600

5

0

4

0

0

0

0

0

0

0

0

0

0

0

0

80

5

2000

5

2

2

0

1

0

0

0

0

0

0

0

0

0

0

100

Female

6

655

5

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

7

819

5

0

1

0

0

0

0

0

0

0

0

0

0

0

0

20

8

1024

5

0

3

0

0

0

0

0

0

0

0

0

0

0

0

60

9

1280

5

1

4

0

0

0

0

0

0

0

0

0

0

0

0

100

10

1600

5

2

3

0

0

0

0

0

0

0

0

0

0

0

0

100

                               LD50 (mg/kg)    95% Confidence limit

Male                     910                        (570 – 1452)

Female                972                        (814 – 1162)

Interpretation of results:
harmful
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
LD50 calculated from the mortalities was 910 mg/kg (95% confidence limit: 570-1452 mg/kg) in male, and 972 mg/kg (95% confidence limit: 814-1162 mg/kg) in female.
On the basis of the results noted in the study, classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008 is as follows:

R22: Harmful if swallowed
H302: Harmful if swallowed
Executive summary:

Acute oral toxicity study of 6-tert-butyl-2,4-xylenol was conducted using 5 male and 5 female CD (SD) rats per each group.

6-tert-butyl-2,4-xylinol was dissolved in corn oil. Single oral administration was made at the dose levels of 819, 1024, 1280, 1600 and 2000 mg/kg for male, 655, 819, 1024, 1280 and 1600 mg/kg for female. During the observation period of 14 days, mortality, toxic signs, and the time of toxic sign appearance were observed. The results of the observations were summarized as follows.

 

LD50: Death of animals was seen at and above dose levels of 1024 mg/kg in male, and 819 mg/kg in female. All male animals treated with 2000 mg/kg died and all females treated with 1280 mg/kg died. LD50was 910 mg/kg (95% confidence limit: 570 to 1452 mg/kg) in male, 972 mg/kg (95% confidence limit: 814 to 1162 mg/kg) in female.

 

Clinical observations: Decrease in locomotive activity was seen at 1 hour until 5 days after the administration. In addition, prone position and hunchback position were observed from 1 hour until 4 days after the administration, and abnormal gait was seen from 6 hours until 3 days after the administration. Hypothermia was observed from 6 hours until 4 days after the administration. In died animals lateral position was seen from 5 hours after the administration and brownish urine was observed from 2 days after the administration both in males and females.

 

Body weight: Body weights of survived animals both in male and female were normally increased until the end of the observation period.

 

Pathological examination

Macroscopic findings: In dead animals, hydrothorax, black patch in the thymus, black patch and retention of brownish urine in the urinary bladder, white patch in the forestomach, black patch in the glandular stomach, white, brown or black contents in the small intestine, white patch in the small intestine and white contents in the large intestine were observed. In the animals survived until the end of observation period, yellow patch in the liver, adhesion with the liver and the spleen, diaphragm, or retro-peritoneum, atrophy of the spleen, adhesion with the spleen and the kidney, thickening of the forestomach, and adhesion with the forestomach and the liver or the diaphragm were observed.

 Histological findings: In the survived male animals at the end of the observation period, severe ulceration with granulomatous inflammation, and hyperplasia of the stratified squamous epithelium with granulomatous inflammation in the forestomach, and granulomatous inflammation in the liver were observed.

 

On the basis of the results noted in the study, classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008 is as follows:

R22: Harmful if swallowed

H302: Harmful if swallowed

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
910 mg/kg bw
Quality of whole database:
K1; GLP studies.

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
07 December 1992 to 26 January 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD test guidelines in compliance with GLP. Although tested in one group only, the test is considered conclusive for classification and labelling.
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
yes
Principles of method if other than guideline:
At the request of the sponsor, the study was finished after the examination of one dose group.
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species: rabbit
Strain: white New Zealands (WNZ)
Source: Harald Schriever, Kaninchenfarm, 2740 Bremervönle, Neuendamm 88
Date of receipt: December 1, 1992 and January 5, 1993
Acclimation period: at least 5 days
Animal selection: random
Animal identification: with individual ear tags; cage labelled with ear tag no., dosage, sex, date of study initiation, project no.
Weight range at study initiation: f: 2.00 - 2.90 kg
Housing: individual housing (50 x 45 x 40 cm, L x B x H) in a battery of cages, each equipped with a paper roll disposal system
Illumination: artificial lighting (120 lux) from 7.00 a.m. - 7.00 p.m.
Temperature: 20 ± 3 °c
Relative humidity: 30 - 70 %
Measurement: with thermohygrometer twice daily
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
Prior to study initiation, the animals were acclimated to laboratory conditions for at least 5 days. Only healthy animals were used in the test.
24 h before treatment, the fur was removed with electric clippers from an area of roughly 8 x 15 cm on the back of each animal. The skin was subsequently examined for abrasions and animals with healthy, intact skin were used in the test.

Preparation and application of the test article: A single dermal application of the test article was performed. The substance was held in contact with the skin with a porous gauze dressing and ElastoplastR (Beiersdorf AG, Hamburg), which was covered with rubber dental dam (4 D Rubber Co., Ltd., UK.) and StülpaiS (P. Hartmann AG, Heidenheim-Brenz).
Duration of exposure:
24 hours
Doses:
The test article was applied undiluted in a volume of 0.21 ml/kg (200 mg/kg).
No. of animals per sex per dose:
Main study - 5 female rabbits
Preliminary study - 2 female & 1 male rabbit
Control animals:
not specified
Details on study design:
Range finding: A preliminary range finding test with doses of 2000, 1000 and 200 mg/kg body weight was conducted on two female rabbits. Another male rabbit was also treated with the test article in a dose of 200 mg/kg.

Main study
Clinical observations: In each animal a number of clinical-toxicological signs were evaluated according to a modified Irwin Screening procedure (Screening Methods in Pharmacology, R. A.
Turner, 1965, p. 26). Any change from the normal condition was noted (increase or decrease) and the degree of severity of any clinical symptoms was assessed. The animals were examined at the following post-treatment intervals: 10 min, 1 h, 2 h, 6 h, 24 h, and thereafter once daily up to day 14.
Body weights: The body weights of all animals were recorded immediately before treatment (day 0) and the surviving animal was reweighed on days 7 and 14 p.a. (termination).
Necropsy: Animals found dead were immediately necropsied. The surviving animal was sacrificed by T 61 injection after 14 days and a gross pathological examination was subsequently performed.
Preliminary study:
There were 6 deaths in the preliminary study.
Sex:
female
Dose descriptor:
LD50
Effect level:
< 200 mg/kg bw
Based on:
test mat.
Mortality:
4 of 5 animals died pre-terminally
Clinical signs:
other: Anmals no. 9 and 10 showed slight abnormal clinical signs such as an increased respiratory rate and/or a reduced activity. In animals no. 6, 7 and 8 no test article dependent findings were observed.
Gross pathology:
Gross pathological examinations in animals found dead and at 14 days p.a. (terminal necropsy) revealed no test article-dependent findings apart from some animals which had serous-serosangineous nasal discharge.
Other findings:
No signs of erythema or oedema were observed.

APPENDIX 2 – CLINICAL OBSERVATIONS

Test article: Lowinox 624-98,5                    Project no.: 10-03-1635/01-92

Species: Rabbit                                            Sex: female

Figures in parentheses indicate number of surviving animals

Dose mg/kg

Observation period p.a.

Specific Findings

200

10 min – 6 h (5/5)

No abnormal clinical signs

24 h (3/5)

Slight increased respiratory rate as well as no abnormal clinical signs

48 h (2/5)

Increased respiratory rate and slight reduced activity as well as no abnormal clinical signs

Days 3 – 14 (1/5)

No abnormal clinical signs

 

APPENDIX 3 – BODY WEIGHTS

INDIVIDUAL VALUES (kg)

Dose: 200 mg/kg

Anim. No.

Sex

Day 0

Day 7 p.a.

Day 14 p.a.

6

F

2.85

2.81

2.93

7

F

2.36

+

+

8

F

2.52

+

+

9

F

2.90

+

+

10

F

2.72

+

+

MEAN VALUES (kg)

Sex

N

Day 0

Day 7 p.a.

Day 14 p.a.

Females

5

2.67

2.81

2.93

 

APPENDIX 5 - INDIVIDUAL VALUES OF SKIN REACTIONS

Anim. No.

Sex

Days p.a.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Ery

Oed

Ery

Oed

Ery

Oed

Ery

Oed

Ery

Oed

Ery

Oed

Ery

Oed

Ery

Oed

Ery

Oed

Ery

Oed

Ery

Oed

Ery

Oed

Ery

Oed

Ery

Oed

6

F

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

7*

F

0

0

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

8*

F

0

0

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

9*

F

0

0

0

0

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

10*

F

0

0

0

0

0

0

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Ery= Erythema

Oed = Oedema

* = Pre-terminal death

 

APPENDIX 6 – NECROPSY

Test article: Lowinox 624-98,5                    Project no.: 10-03-1635/01-92

Species: Rabbit                                           Animal no.: 6 – 10

Sex: Female                                                 Dose: 200 mg/kg

Spontaneous death: 24 h (no. 7, 8), 48 h (no. 9), 3 days p.a. (no. 10)        n = 4

Killed in extremis: ----                                                                               n = -

Animal no.

Specific Findings

7

Serous nasal discharge

10

Serosanguineous nasal discharge

8, 9

No specific findings

Terminal sacrifice: 14 days p.a.                  n= 1

Animal no.

Specific Findings

6

No specific findings

Interpretation of results:
toxic
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
LD50 < 200 mg/kg in rabbits.

On the basis of the results noted in the study, classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008 is as follows:

R27: Very toxic in contact with skin.
H310: Fatal in contact with skin
Executive summary:

The aim of the test was to determine the acute median lethal dose of "Lowinox 624-98,5". Information derived from the test serves to indicate the existence of possiblehazards likely to arise from short-term exposure to the test article by the dermal route.

The test was conducted according to the OECD guideline for the testing of chemicalsOECD 402 (February 24, 1987) and to the EEC diective 84/449ÆEC (September 19,1984). The study was carried out as described in the corresponding protocol approved by the testing facilty and the study sponsor on November 13 and November 17, 1992. The principles of Good Laboratory Practice for the testing of chemicals as specified bynational (BGBL. I, no 13, § 19 a, March 22, 1990) and international (OECD, Paris,1982) legislation were followed during the performance ofthe study.

 

The acute dermal toxicity of "Lowinox 624-98,5" was investigated in 5 female rabbits.

On the basis of the range finding results, each animal was given a single dermal administration of "Lowinox 624-98,5" at a dose of 200 mg/kg body weight. Theskin was exposed to the test article for 24 h and subsequently evaluated once daily for14 days.

Clinical observations were conducted at regular intervals during the 14-day observation period. Body weights were measured at day 0 and in animal no. 7 at days 7 and 14 p.a.Gross pathological examinations were performed in animals found dead and at 14days p.a.

The following results were obtained in the main study:

1. 4 of 5 animals died pre-terminally

2. Two animals (no. 9 and 10) showed only slight abnormal clinical signs such as an increased respiratory rate and/or a reduced activity.

3. Weight gain was reduced in the surviving animal at days 7 and 14 p.a.

4. Gross pathological examinations in animals found dead and at 14 days p.a.(terminal necropsy) revealed no test article-dependent findings, apart from twoanimals (no. 7 and 10) which had serious or serosanguineous nasal discharge.

5. The following LD50 value was determined after 24h and 14 days:

                               24h                       14 days

Female               > 200 mg/kg       < 200 mg/kg

 

At the request of the sponsor, the study was finished after the examination of one dose group.

On the basis of the results noted in the study, classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008 is as follows:

R27: Very toxic in contact with skin.

H310: Fatal in contact with skin

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
50 mg/kg bw
Quality of whole database:
K1; GLP studies. Lowest value available for the rabbit study is taken, as the key study gives LD50 at <200 mg/kg.

Additional information

Oral

Two studies are available for review for this endpoint. 

 

Key study:

In the key study, acute oral toxicity study of 6-tert-butyl-2,4-xylenol was conducted using 5 male and 5 female CD (SD) rats per each group. Single oral administration was made at the dose levels of 819, 1024, 1280, 1600 and 2000 mg/kg for male, 655, 819, 1024, 1280 and 1600 mg/kg for female. During the observation period of 14 days, mortality, toxic signs, and the time of toxic sign appearance were observed. The results of the observations were summarized as follows.

 

LD50: Death of animals was seen at and above dose levels of 1024 mg/kg in male, and 819 mg/kg in female. All male animals treated with 2000 mg/kg died and all females treated with 1280 mg/kg died. LD50was 910 mg/kg (95% confidence limit: 570 to 1452 mg/kg) in male, 972 mg/kg (95% confidence limit: 814 to 1162 mg/kg) in female.

 

Clinical observations: Decrease in locomotive activity was seen at 1 hour until 5 days after the administration. In addition, prone position and hunchback position were observed from 1 hour until 4 days after the administration, and abnormal gait was seen from 6 hours until 3 days after the administration. Hypothermia was observed from 6 hours until 4 days after the administration. In died animals lateral position was seen from 5 hours after the administration and brownish urine was observed from 2 days after the administration both in males and females.

 

Body weight: Body weights of survived animals both in male and female were normally increased until the end of the observation period.

 

Pathological examination

Macroscopic findings: In dead animals, hydrothorax, black patch in the thymus, black patch and retention of brownish urine in the urinary bladder, white patch in the forestomach, black patch in the glandular stomach, white, brown or black contents in the small intestine, white patch in the small intestine and white contents in the large intestine were observed. In the animals survived until the end of observation period, yellow patch in the liver, adhesion with the liver and the spleen, diaphragm, or retro-peritoneum, atrophy of the spleen, adhesion with the spleen and the kidney, thickening of the forestomach, and adhesion with the forestomach and the liver or the diaphragm were observed.

 

Histological findings: In the survived male animals at the end of the observation period, severe ulceration with granulomatous inflammation, and hyperplasia of the stratified squamous epithelium with granulomatous inflammation in the forestomach, and granulomatous inflammation in the liver were observed.

 

Supporting study

Following a range-finding study, a group of ten fasted animals (five males and five females) was given a single oral dose of undiluted test material at a dose level of 2000 mg/kg bodyweight. An additional group of ten fasted animals was then treated with the test material administered as a solution in arachis oil B.P. at a dose level of 200 mg/kg bodyweight. The surviving animals were observed for 14 days after the day of dosing. All animals were subjected to gross pathological examination.

The acute median lethal dose (LD50) of the test material in the SpragueDawley strain rat was found to be greater than 200 mg/kg bodyweight but less than 2000 mg/kg bodyweight.

 

On the basis of the results noted in the study, classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008 is as follows:

R22: Harmful if swallowed

H302: Harmful if swallowed

 

Inhalation.

 

No data available. The substance is of low volatility and is not utilised in exercises where dusts or aerosols could be generated. As a result, inhalation is not considered to be a primary route of exposure.

 

Dermal

 

Four studies are available for this endpoint. As follows:

 

Rabbit – Study 1

 

The acute dermal toxicity of the substance was investigated in 5 female rabbits.

On the basis of the range finding results, each animal was given a single dermal administration of the substance at a dose of 200 mg/kg body weight. The skin was exposed to the test article for 24 h and subsequently evaluated once daily for14 days.

 

Clinical observations were conducted at regular intervals during the 14-day observation period. Body weights were measured at day 0 and in animal no. 7 at days 7 and 14 p.a.Gross pathological examinations were performed in animals found dead and at 14days p.a.

The following results were obtained in the main study:

 

1. 4 of 5 animals died pre-terminally

2. Two animals (no. 9 and 10) showed only slight abnormal clinical signs such as an increased respiratory rate and/or a reduced activity.

3. Weight gain was reduced in the surviving animal at days 7 and 14 p.a.

4. Gross pathological examinations in animals found dead and at 14 days p.a.(terminal necropsy) revealed no test article-dependent findings, apart from twoanimals (no. 7 and 10) which had serious or serosanguineous nasal discharge.

5. The following LD50 value was determined after 24h and 14 days:

                               24h                       14 days

Female               > 200 mg/kg       < 200 mg/kg

 

At the request of the sponsor, the study was finished after the examination of one dose group.

 

Rabbit – Study 2

A study was performed to assess the acute dermal toxicity of the test material in the New Zealand White rabbit.

Following a range-finding study, a group of ten animals (five males and five females) was given a single 24-hour, semi-occluded dermal application to intact skin at a dose level of 50 mg/kg bodyweight. The surviving animals were observed for fourteen days after the day of treatment and were then killed for gross pathological examination.

Three males and one female were found dead two days after dosing. One male and three females were killed in extremis one or two days after dosing. Common signs of systemic toxicity noted were hunched posture, lethargy, decreased respiratory rate, laboured respiration and increased salivation with additional signs or isolated incidents of ataxia, dehydration, pallor of the extremities, hypothermia, ptosis, gasping, increased or noisy respiration, loss of righting reflex and red/brown staining around the snout.

Surviving animals showed an expected gain in bodyweight during the study.

Haemorrhagic lungs were noted at the necropsy of all animals that died during the study with additional abnormalities of dark liver, pale spleen, haemorrhage or slight haemorrhage of the gastric mucosa, epithelial sloughing of the gastric mucosa, haemorrhage or slight haemorrhage and epithelial sloughing of the non-glandular region of the stomach. No abnormalities were noted in the animals that were killed at the end of the study.

The acute dermal median lethal dose (LD50) of the test material in the New Zealand White rabbit was found to be less than 50mg/kg bodyweight.

 

Rat – Study 1

 

The acute dermal toxicity of the substance was investigated in 5 male and 5 female Wistar rats. On the basis of the range finding results, each animal was given a single dermal administration of the substance at a dose of 2000 mg/kg body weight. The skin was exposed to the test article for 24 h and signs of erythema and oedema were subsequently evaluated once daily for 14 days.

Clinical observations were conducted at regular intervals during the 14-dayobservation period. Body weights were measured at days 0, 7 and 14 p.a. Gross pathological examinations were performed on animals at termination.

The following results were obtained:

 

1. No abnormal clinical signs were observed.

2. No signs of oedema were observed. At day 1 p.a. very slight to well-defined erythema were observed. At days 4 and 5 p.a. in two females very slight erythema and slight scale formation were observed.

3. Weight gains were reduced in most ofthe animals at day 7 p.a. In one male and in one female the body weight was decreased at day 7 p.a. At day 14 p.a. weight gains were normal in the males but reduced in the females.

4. Gross pathological examinations at 14 days p.a. (terminal necropsy) revealedno test article-dependent findings.

5. According to the requirements of the limit test, the LD50 values after 24 h and 14 days were as follows:

male and female> 2000 mg/kg

 

When applied to the skin of the rat, the substance was deemed to be "non-toxic".

No classification and labelling is applicable.

 

Rat – Study 2

 

The acute dermal toxicity of the substance in a 55% mixture was investigated in 5 male and 5 female Wistar rats. On the basis of the range finding results, each animal was given a single dermal administration of the substance at a dose of 2000 mg/kg body weight. The skin was exposed to the test article for 24 h and signs of erythema and oedema were subsequently evaluated once daily for 14 days.

 

The following results were obtained:

 

1. No abnormal clinical signs were observed apart from a slightly decreased skin turgor at 24 h p.a.

 

2. Very slight to well-defined erythema was observed in most animals until day 8 p.a. and in 4 animals until day 12 p.a. Very slight oedema was observed at day 1 p.a., in one female at the days 3, 4, 5 p.a. and in 3 females at day 6 p.a. Additionally, formation of cracks, scaliness, induration and peeling of the injured skin were observed between days 3 and 12 p.a.

 

3. Weight gains were normal in all animals apart from a decreased body weight in one female at day 7p.a.

 

4. Gross pathological examinations at14 days p.a. (terminal necropsy) revealed no test article-dependent findings.

 

According to the requirements of the limit test, the LD50 values after 24 h and 14 days were as follows:

male and female>2000 mg/kg

 

When applied to the skin of the rat, the substance was deemed to be "non-toxic".

No classification and labelling is applicable.

 

Discussion

 

It was noted that there is a large distinction between the results noted in the rat and the rabbit. Rats are largely unaffected by the substance, whereas rabbits demonstrate a toxic response to the substance when applied dermally.  The studies carry no definitive explanation of this difference of toxicity observed, and it is concluded that this is a species specific effect. 

Assessment of the literature, specifically:

Skin Permeability In Vivo: Comparison in Rat, Rabbit, Pig and Man;Journal of Investigative Dermatology(1972)58, 114–123; doi:10.1111/1523-1747.ep12538909 Accepted 26 October 1971.

 

This paper details that in a comparative percutaneous absorption study undertaken in rats, rabbits, miniature swine and man, the results obtained indicated that skin permeability decreases in the following order: rabbit, rat, pig and man. It may be therefore that the permeability of rabbit skin is much greater for the substance than it is in the rat, and the toxic response is proliferated by rabbit skin to a greater extent.

 

On the basis of the rabbit results, it is deemed appropriate to consider the classification and labelling on a worst case basis. As a result, the rabbit results are taken as the definitive value.On the basis of the results noted in the study, classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008 is as follows:

 

Acute toxicity, Category 1

H310: Fatal in contact with skin

R27: Very toxic in contact with skin.

Justification for selection of acute toxicity – oral endpoint

Study performed in accordance with OECD test guidelines No. 401.  Two studies are available to address this endpoint; the definitive lowest LD50 value is taken as the key value, for the purposes of hazard assessment.  Both studies are in agreement with regards to classification and labelling.

Justification for selection of acute toxicity – inhalation endpoint

No data available. The substance is of low volatility and is not utilised in exercises where dusts or aerosols could be generated.  As a result, inhalation is not considered to be a primary route of exposure.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP and the results are well documented. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

 

The above results triggered classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). The following classification for acute effects is therefore required:

 

H302: Harmful if swallowed

H310: Fatal in contact with skin

 

R22: Harmful if swallowed

R27: Very toxic in contact with skin.